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Virus capping system polypeptide inhibitor for controlling coronavirus

A coronavirus, sequence technology, applied in the direction of antiviral agents, medical preparations containing active ingredients, peptides, etc.

Inactive Publication Date: 2012-09-19
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

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  • Virus capping system polypeptide inhibitor for controlling coronavirus
  • Virus capping system polypeptide inhibitor for controlling coronavirus
  • Virus capping system polypeptide inhibitor for controlling coronavirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] The design and verification of embodiment 1 polypeptide inhibitor

[0022] 1. Crystal structure analysis and inhibition mechanism of SARS coronavirus nonstructural protein nsp10 and nsp16 complex

[0023] figure 1 Shown is a diagram of the crystal structure of the co-crystal of the nsp10 and nsp16 complex. figure 1 In A, the green one is nsp10, and the light blue one is nsp16. It can be seen that the two interact through the protein surface to form a complex. According to our previous studies, nsp10 promotes the binding of nsp16 to the substrate (viral RNA) and the binding to the substrate SAM, thereby activating the 2'-O-MTase methyltransferase activity of nsp16. figure 1B shows the surface charge distribution map of nsp16, where the blue area is the positively charged surface (such as the area indicated by arrow 1), and the red area is the negatively charged surface (such as the area indicated by arrow 2). Since the viral RNA is negatively charged, it can bind to t...

Embodiment 2

[0036] Example 2: Polypeptide inhibitor K29 inhibits the combination of SARS coronavirus non-structural protein nsp16 and substrate SAM

[0037] Reagent preparation:

[0038] 10×Reaction buffer: 400mM Tris-HCl (pH 7.5), 20mM MgCl 2 , 20mMDTT.

[0039] isotope 3 H substrate: S-adenosyl[methyl-3H]methionine (67.3 Ci / mmol, 0.5 μCi / μl).

[0040] Display enhancer: Enlightening buffer (PerkinElmer).

[0041] Implementation steps:

[0042] 1. In a 25μl reaction system [40mM Tris-HCl (pH 7.5), 2mM MgCl 2 , 2mM DTT] added the purified SARS coronavirus non-structural proteins nsp16 (0.5 μM) and nsp10 (4 μM) (Chen et al., 2011).

[0043] 2. Add different concentrations of peptide inhibitor K29 (0 μM, 10 μM, 100 μM, 400 μM) or control polypeptide K8 (0 μM, 10 μM, 100 μM, 500 μM) into the reaction, and mix well.

[0044] 3. Add 2μCi S-adenosyl[methyl-3H]methionine (67.3Ci / mmol, 0.5μCi / μl) to each reaction system and mix well.

[0045] 4. Place the reaction system on ice, and irradi...

Embodiment 3

[0050] Embodiment 3: polypeptide inhibitor K29 and K12 suppress the methyltransferase activity (viral RNA capping system activity) of SARS coronavirus nonstructural protein nsp16

[0051] Reagent preparation:

[0052] 10×Reaction buffer: 400mM Tris-HCl (pH 7.5), 20mM MgCl 2 , 20mMDTT, 400units RNase inhibitor, 0.1mM SAM.

[0053] isotope 3 H substrate: S-adenosyl[methyl-3H]methionine (67.3Ci / mmol, 0.5μCi / μl)

[0054] Viral RNA substrate: m7GpppA-RNA (derived from SARS coronavirus RNA)

[0055] RNA purification medium: activated DEAE-Sephadex G250

[0056] Implementation steps:

[0057] 1. In a 30μl reaction system [40mM Tris-HCl (pH 7.5), 2mM MgCl 2 , 2mM DTT, 40units RNase inhibitor, 0.01mM SAM] added the purified SARS coronavirus nonstructural proteins nsp16 (3.3μM) and nsp10 (14μM).

[0058] 2. Add different concentrations (0-320 μM) of peptide inhibitors K29 or K12, and other control peptides K8, K10, and K20 to the reaction and mix well.

[0059] 3. Add 2μCi S-ade...

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Abstract

The invention relates to a virus capping system polypeptide inhibitor for controlling coronavirus and a method for inhibiting the activity of the coronavirus capping system by utilizing the polypeptide inhibitor. The polypeptide inhibitor comprises the polypeptide with the following amino acid sequence: 1) an amino acid sequence shown in SEQ ID No.1, 2) a sequence which has equivalent functions, is derived from the sequence shown in SEQ ID No.1 and is obtained by replacing, adding and / or deleting one or more amino groups in the amino acid sequence shown in SEQ ID No.1, or 3) a truncated sequence of the sequence shown in SEQ ID No.1, wherein the truncated sequence at least comprises GGASCCLYCRCH. The inhibitor has extremely high specificity, overcomes the defect that the traditional virus capping system inhibitors have low specificity and simultaneously overcomes the defect that the present nonspecific treatment drugs have high side effects and are easy to cause sequelae as a guide drug for controlling coronavirus.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, virology, biochemistry, protein (enzyme) structure and function research and biomedicine. In particular, it relates to a polypeptide inhibitor that can be used to inhibit the capping modification function and its replication and transcription of coronaviruses such as SARS-CoV. Background technique [0002] Coronavirus (Coronavirus) is a kind of pathogenic RNA virus that infects humans, poultry, and livestock, and then causes respiratory tract, digestive tract inflammation, and diarrhea. Among them, severe acute respiratory syndrome (Severe Acute Respiratory Syndromes, referred to as SARS) caused by SARS coronavirus (SARS-CoV) infection is a new respiratory infectious disease, with fever, dry cough, and chest tightness as the main symptoms. Rapidly progressive respiratory failure, highly contagious and rapidly progressive (Ziebuhr, 2003). In 2003, when the virus broke out worldwide, th...

Claims

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Application Information

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IPC IPC(8): C07K14/00C07K7/08A61K38/16A61K38/10A61P31/14C12N15/11C12N15/63C12N1/21A61K48/00
Inventor 郭德银陈宇柯敏吴安东
Owner WUHAN UNIV
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